Little is known about availability of cholesterol for absorption from foods, experimental formulations and mixed intestinal contents. To examine the variability of cholesterol bioavailability, we measured absorption of various physiochemical forms of cholesterol in normal human subjects during test meals. Pentadeuterated and hexadeuterated cholesterol tracers were employed and enrichment of plasma cholesterol was measured by gas chromatography/negative ion chemical ionization mass spectrometry. Since most dietary cholesterol is complexed with phospholipid, percent cholesterol absorption was measured in 6 subjects on two occasions, once with cholesterol tracer in corn oil and once with tracer in egg yolk phospholipid. Cholesterol absorption was similar during both tests (53.6(3.6% with corn oil and 56.5(3.9% with phospholipid). Solubilization of the cholesterol in a lipophilic medium apparently was not necessary since solid anhydrous cholesterol was absorbed wit h the sam e efficiency as cholesterol completely dissoved in corn oil when given to 4 subjects in a single test meal (mole ratio in plasma/mole ratio administered 0.998(0.087). However, when microcrystalline cholesterol monohydrate and anhydrous cholesterol powders were mixed and given in a capsule to 6 subjects the mole ratio of cholesterol monohydrate/anydrous cholesterol absorbed was reduced to 0.67(0.13 (p=0.03). These results show that three anhydrous forms of cholesterol were equally well absorbed. However, the absorption of cholesterol monohydrate was significantly lower and its potential role in human cholesterol absorption and metabolism needs further study.